A digestive β-glucosidase from the salivary glands of the termite, Neotermes koshunensis (Shiraki): distribution, characterization and isolation of its precursor cDNA by 5′- and 3′-RACE amplifications with degenerate primers
Introduction
Termites play a significant role in maintaining carbon balance in nature, and it has long been considered that cellulosic materials are primarily digested by the symbiotic protozoa in their hindguts. Cellulose is decomposed by three kinds of enzymes, which are cellobiohydrolase (EC. 3.2.1.91), endo-β-1,4-glucanase (EC. 3.2.1.4) and β-glucosidase (EC. 3.2.1.21). We have previously shown that termites themselves produce their own endo-β-1,4-glucanase, which hydrolyze cellulose primarily to cellobiose (Watanabe and Tokuda, 2001). Cellobiose and other cello-oligosaccharides are degraded to glucose by β-glucosidase, and therefore it is important to study β-glucosidase to aid understanding of the digestive physiology of termites.
Apart from termites, β-glycosidase has been studied in several insects (Terra et al., 1996). Insect β-glycosidases are classified into three classes based on their substrate specificities, and two (Class 1 and Class 2) of the three classes show typical β-glucosidase activity. Class 1 enzymes are active not only on cellobiose or lactose but also synthetic substrates such as p-nitrophenyl-β-D-glucopyranoside and p-nitrophenyl-β-D-fucopyranoside, whereas Class 2 enzymes can only hydrolyze cellobiose and lactose (Terra et al., 1996). Because most insects do not feed on cellulose, these enzymes are considered to play a role in completing hemicellulose digestion (Terra et al., 1996).
Termites (order Isoptera) consist of seven families. It is known that termites belonging to the family Termitidae do not have cellulolytic protozoa in their guts, while those of the other families possess numerous symbiotic flagellates in their hindguts (Inoue et al., 2000). Phylogenetic studies suggest that the family Termitidae is the most recent lineage of Isoptera and that the family Rhinotermitidae shows a much closer relationship to the family Termitidae than the other basal families (Kambhampati and Eggleton, 2000, Thompson et al., 2000). Recent molecular and biochemical studies of endo-β-1,4-glucanase have indicated that the salivary glands play a significant role in the cellulose digestion of not only termites belonging to the family Rhinotermitidae (Watanabe et al., 1997, Watanabe et al., 1998, Tokuda et al., 1999) but also those derived from other lineages, including Kalotermitidae (Lo et al., 2000, Thompson et al., 2000). Regarding β-glucosidase, biochemical studies have often indicated that the midgut has a large amount of activity in termites (Slaytor, 2000), but a possible role for the salivary glands has not been considered. In addition, no studies have yet been performed with respect to molecular characterization of β-glucosidase in termites. Thus, it is valuable to elucidate the expression site and enzymatic characteristics of endogenous (termite-origin) β-glucosidase, which is likely to be an indispensable part of the cellulose digesting system of termites.
In the present paper, we describe biochemical and molecular characterization of a salivary β-glucosidase from the termite, Neotermes koshunensis [Isoptera: Kalotermitidae].
Section snippets
Termites
Logs inhabited by the termite Neotermes koshunensis (Shiraki) [Isoptera: Kalotermitidae], were collected on Okinawa-island, located in the subtropical region of Japan. The termites were kept at room temperature with nest materials. Worker-caste termites (pseudoworkers) were utilized throughout the investigations.
Distribution of β-glucosidase activity in the gut
For preparation of enzyme extracts, the salivary glands and whole guts were removed from ten termites and divided into the foregut, midgut and hindgut. The salivary glands and each of
β-Glucosidase activity in the gut
Table 1 shows the distribution of β-glucosidase activity from the salivary glands to the hindgut of N. koshunensis. The highest activity was detected in the salivary glands, while 15% of the activity was found in the hindgut. The specific activity was also highest in the salivary glands. However, no significant difference in the specific activity was observed throughout the different parts of the gut.
Partial purification of the β-glucosidase
As the major part of β-glucosidase activity was the salivary glands, we dissected 500 termites
Discussion
Recent progress in molecular studies of cellulases has improved our understanding of cellulose digestion in the animal kingdom (Watanabe and Tokuda, 2001). In termites, several endo-β-1,4-glucanase cDNAs have been sequenced and their origins clarified (Watanabe and Tokuda, 2001), but similar research on digestive β-glucosidases has not been performed, despite the important role played by these enzymes during termite cellulose digestion (Watanabe et al., 1997, Tokuda et al., 1997, Nakashima et
Acknowledgements
We thank Dr. K. Nakashima for critical reading of an earlier draft and Dr. N. Lo for comments on the manuscript and for English correction. This work was supported by the Promotion of Basic Research Activities for Innovative Biosciences Fund from the Bio-oriented Technology Research Advancement Institution (BRAIN; Omiya, Saitama, 331-8537 Japan; www.brain.go.jp) and the Pioneer Research Project Fund (No. PRPF-0022) from the Ministry of Agriculture, Forestry and Fisheries of Japan to H.W, and by
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